1. Field of the Invention
The present invention relates to a novel pharmaceutical composition for the treatment of ischemia, which contains a conjugate of a molecule of interest, such as a phospholipase polypeptide, and a protein transduction domain (PTD), as well as a method for delivering the same.
2. Background Art
The body is critically dependent on the heart to pump blood. A healthy heart pumps blood throughout the body for the delivery of oxygen and nutrients and the removal of harmful products of metabolism. Ischemia leads to rapid changes in myocardial metabolism and cardiac and cellular injury. The extent of the injury is dependent on the severity of ischemia and the timeliness of appropriate treatment. Continued ischemia can lead to total tissue necrosis in a few hours.
Reperfusion, although generally considered beneficial, causes tissue injury by several mechanisms. Clinically, in open heart surgery, heart transplantation, and reversal of heart disease, protection of the myocardium against injury by ischemia-reperfusion is an issue of utmost clinical interest. Exacerbation of hypoxic injury after restoration of oxygenation (reoxygenation) by reperfusion is an important mechanism of cellular injury in other types of organ transplantation and in hepatic, intestinal, cerebral, renal, and other ischemic syndromes.
Ischemia and simulated ischemic conditions cause an increase in active oxygen species and an overload of calcium ions (Ca2+) (Bolli, R., et al., Physiol. Rev. 79:609-634 (1999)). Cytosolic calcium accumulation has been proposed as a mediator of pathologic changes that occur during myocardial ischemia (Moraru, I. I., et al., Biochim. Biophys. Acta 1268:1-8 (1995)). The increase in intracellular calcium results in the opening of mitochondrial permeability transition pores (mPTPs). The increase in intracellular calcium further enhances the opening of additional mPTPs and also activates a number of cytosolic proteins, such as phospholipases, protein kinases, proteases, and endonucleases (Bolli, R., et al., Physiol. Rev. 79:609-634 (1999)). It has been reported that when treatment with mPTP inhibitors cyclosporin A and sanglifehrin A is performed in post-ischemic reperfusion, the recovery of systolic function and the viability of cells increases by about 20% and 62%, respectively (Javadov, S. A., et al., J. Physiol. 549:513-524 (2003)).
Phospholipases, such as phospholipase C (PLC), play an important role in the regulation of calcium homeostasis. To date, eleven mammalian PLC isozymes have been identified. These can be divided into four types: PLC-β, PLC-γ, PLC-δ and PLC-ε. PLC δ1 and γ1 are the predominant forms in normal cardiac cells (Hansen, C. A., et al., J. Mol. Cell. Cardiol. 27:471-484 (1995); and Schnabel, P., et al., J. Mol. Cell. Cardiol. 28:2419-2427 (1996)).
All PLC isozymes contain a C2 domain that is sensitive to Ca2+ activation (Hwang, K-C, et al., J. Steroid Biochem. 91:131-138 (2004)). Among the PLC isoforms, PLC-δ1 is most sensitive to activation by intracellular Ca2+. Id.
PLC hydrolyzes the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) (Hwang, K-C, et al., J. Steroid Biochem. 91:131-138 (2004)). DAG and IP3 stimulate the activity of protein kinase C (PKC) and the release of calcium ions from intracellular reservoirs to the cytoplasm. Id. The activation mechanism of PLC is well known through PLC-β which is activated by a G-protein-coupled receptor, receptor tyrosine kinase, and ras pathway, respectively (Rhee, S. G., et al., Annu. Rev. Biochem. 70:281-312 (2001)).
Based on recent studies, it was reported that PLC-δ1 present in the mitochondrial membrane of liver cells functions to inhibit the inflow of calcium, when an excess of calcium is present in the cytoplasm (Hwang, K-C, et al., J. Steroid Biochem. 91:131-138 (2004)). It was further shown that PLC-δ1 is present in normal myocardial cells in amounts at least 7 times greater than that of the other isozymes, and that, in an ischemic state, the amount of PLC-δ1 decreases both in vitro and in vivo (Hwang, K-C, et al., Steroid Biochem. 91:131-138 (2004)). When treated with the calpain inhibitor calpastatin and the caspase inhibitor zVAD-fmk, the degradation of PLC-δ1 was inhibited. Id. In addition, when PLC-δ1 was overexpressed in cardiomyocytes, intracellular Ca2+ overload induced by ischemic conditions was dramatically rescued. Id.
These results demonstrate the critical role PLC-δ1 plays in cytosolic calcium homeostasis in normal hearts and its effect on calcium balance after myocardial infarction. Clearly, an effective method of transducing PLC-δ1 into the cytosol and nucleus of living cells to treat or prevent ischemia or ischemic conditions, without deleterious side effects, is needed.
Protein transduction domains (PTDs) have been used for delivery of biologically active molecules (Viehl C. T., et al., Ann. Surg. Oncol. 12:517-525 (2005); Noguchi H., et al., Nat. Med. 10:305-309 (2004); and Fu A. L., et al., Neurosci. Lett. 368:258-62 (2004)). PTDs are low molecular-weight peptides that have been used for the penetration of physiologically active molecules into cells. However, to date no attempts have been made to use PTDs as a way of delivering phospholipase C in vivo.